Preparation of antibiotic LL-D05139β from cultures of Glycomyces harbinensis, gen. nov., sp. nov.

ABSTRACT

This disclosure describes a new antibacterial and anti-tumor agent designated LL-D05139β, produced in a microbiological fermentation under controlled conditions using a new genus Glycomyces harbinensis gen. nov., sp. nov., and mutants thereof.

This is a division of application, Ser. No. 488,496, filed Apr. 25,1983.

BRIEF SUMMARY OF THE INVENTION

This invention relates to a new antibacterial and anti-tumor agentdesignated LL-D05139β, to its production by fermentation, to methods forits recovery and concentration from crude solutions and to processes forits purification. The present invention includes within its scope theantibacterial and anti-tumor agent in dilute form, as a crudeconcentrate and in pure form. The effects of this new agent on specificmicroorganisms, together with its structure and chemical and physicalproperties, differentiate it from previously described antibacterialagents.

LL-D05139β has the structure ##STR1## and its chemical abstractsnomenclature is N-L-alanyl-L-serine diazoacetate.

It is related to, but structurally different from, serine(2-amino-3-hydroxypropionic acid) whose structure is ##STR2## andazaserine (serine diazoacetate) whose structure is ##STR3## which is aknown anti-neoplastic agent.

LL-D05139β is an organic carboxylic acid and thus is capable of formingsalts with nontoxic pharmaceutically acceptable cations. Thus saltsformed by admixture of the antibiotic free acid with stoichiometricamounts of cations, suitably in a neutral solvent, may be formed withcations such as alkali metals (e.g., sodium, potassium, etc.), alkalineearth metals (e.g., calcium, magnesium, etc.) and ammonium. The cationicsalts of LL-D05139β are, in general, water soluble solids relativelyinsoluble in most common organic solvents.

DETAILED DESCRIPTION OF THE INVENTION

The new antibacterial agent designated LL-D05139β is formed during thecultivation under controlled conditions of a new strain of a new genusand species named Glycomyces harbinensis, gen. nov., sp. nov.

Depending on the fermentation conditions, variable amount of azaserineis coproduced. Under proper cultivation conditions, using defined mediumLL-D05139β is produced as the major active component. The physiochemicalcharacteristics of LL-D05139β are as follows:

1. Molecular weight: 244 (FAB-MS).

2. UV_(max) at 250 nm, E_(1cm) ^(1%) =646 (UV spectrum as shown in FIG.I, water solution).

3. IR spectrum (KBr disc) as shown in FIG. II.

4. Optical Rotation: [α]_(D) ²⁶ =+57°±5° (C,0.19%, water).

5. ¹ H NMR Spectrum (D₂ O, ppm from TMS) as shown in FIG. III, 1.56(3H,d, J=7.4 Hz), 4.07(1 H, q. J=7.4 Hz), 4.49(2-3H, m), 5.19(1H, broad s).

6. Decomposes rapidly below pH 5.0, stable in weakly basic solutions.

Acid hydrolysis of LL-D05139 releases one molar equivalent each ofL-alanine and L-serine as determined by standard amino acid analysis.Both alanine and serine derived from LL-D05139 have the L-configurationas determined by the well-accepted gas chromatography procedure usingn-Lauroyl-L-valine-t-butylamide as stationary phase. See R. Charles etal., J. Chromatography, 112: 121-133 (1975). N-Terminal analysis ofLL-D05139β using the Edman degradation procedure followed by HPLCanalysis of the PTH-amino acid identified the N-terminal amino acid tobe L-alanine. Therefore this agent has been assigned the followingstructure: ##STR4##

This new antibiotic producing strain was isolated from a soil samplecollected in Harbin, China and is maintained in the culture collectionof the Lederle Laboratories Division, American Cyanamid Company, PearlRiver, N.Y. as culture number LL-D05139. A viable culture of this newmicroorganism has been deposited with the Culture Collection Laboratory,Northern Utilization Research and Development Division, U.S. Departmentof Agriculture, Peoria, Ill., has been added to its permanent collectionunder the accession number NRRL 15337, and is freely available to thepublic from this depository.

Taxonomic Characterization of Culture LL-D05139

The culture LL-D05139 was taxonomically characterized and identified asthe type species of a new genus to be known as Glycomyces harbinensisgen. nov., sp. nov.

Observations were made of the cultural, physiological and morphologicalfeatures of the culture using methods detailed by E. B. Shirling and D.Gottlieb, Methods for characterization of Streptomyces species.,Internat. J. Syst. Bacteriol. 16: 313-340 (1967), and R. E. Gordon, etal., Nocardia colliaca, Nocardia autotrophica, and the nocardia strain.,Internat. J. Syst. Bacteriol., 24: 54-63 (1974). Media used in thisstudy were selected from those recommended by T. G. Pridham, et al., Aselection of media for maintenance and taxonomic study ofStreptomycetes., Antibiotics Ann., 947-953 (1956-57), G. F. Gauze, etal., Problems in the classification of antagonistic actinomycetes.,State Publishing House for Medical Literature. Medgiz Moscow (1957) andR. E. Gordon, et al., vide supra for the taxonomic study ofactinomycetes and soil bacteria. Chemical composition of the cell wallsof the culture was determined using the method of H. A. Lechevalier, etal., Chemical composition as a criterion in the classification ofActinomycetes., Adv. Appl. Microbiol. 14: 47-72 (1971). Phospholipidpatterns were determined using the method of M. P. Lechevalier, et al.,Chemotaxonomy of aerobic actinomycetes: phospholipid composition.,Biochem. Syst. Ecol., 5: 249-260 (1977). Details are recorded in TablesI-VI, and a general description of the culture is given below.Underscored descriptive colors are taken from K. L. Kelly and D. B.Judd, Color. Universal Language and Dictionary of Names, Nat. Bur.Stand. (U.S.), Spec. Publ. 440, Washington, D.C. (1976) and theaccompanying Inter-Society Color Council, Nat. Bur. Stand. CentroidColor Charts.

The data observed for this culture were compared with data published forknown genera of the Actinomycetales M. Goodfellow, et al., Numericaltaxonomy of Actinomadura and related actinomycetes., J. Gen. Microbiol.,112: 95-111 (1979), T. Hasegawa, et al., New Genus of theActinomycetales: Actinosynnema gen. nov., Internat. J. Syst. Bacteriol.,28 (2): 304-310(1978), and J. Meyer, Nocardiopsis, a new genus of theorder Actinomycetales., Internat. J. Syst. Bacteriol., 26 (4):487-493(1976). Isolate LL-D05139 bears no close resemblance to any ofthe known actinomycete genera, with Nocardiopsis being the most closelyrelated taxon from a morphological standpoint. The whole-cell chemistryof LL-D05139 is Type II-D (meso-DAP, and arabinose and xylose as thecharacteristic sugars). The phospholipid pattern of this culture, asobserved by thin-layer chromatography, represents a very unusual patternfor the Type P-I group. The Type II-D whole cell analysis, usuallytypical of Micromonospora, and the unusual Type P-I phospholipid patternare totally novel for a microorganism possessing the morphology of thisculture. Based on these observations, the culture is assigned to a newgenus designated as Glycomyces. The species name harbinensis refers tothe site in Harbin, China where the soil sample was collected from whichthis culture was isolated.

Micromorphology

Spores are formed in very short straight chains on rudimentary aerialsporophores. The spores are cylindrical in shape, 0.45 to 0.55 micronsby 0.98 to 1.06 microns, and have a smooth surface.

Cell Wall Composition

Whole cell hydrolysates of this culture contain the meso-isomer ofdiaminopimelic acid, arabinose and xylose as the characteristic sugars.The culture has an unusual variation of the Type P-I phospholipidpattern and contains phosphatidyl glycerol, phosphatidyl inositol andphosphatidyl inosityl mannosides. This phospholipid composition has notbeen reported for any other genus in the Actinomycetales to date.

Amount of Growth

Good growth observed on NZ-amine-starch-glucose agar (ATCC Medium 172)and yeast extract-malt extract agar; moderate growth observed onBenedict's agar, Bennett's agar, calcium malate agar, Czapek's sucroseagar, Gauze No. 1 agar, Hickey-Tresner agar, and oatmeal agar; poorgrowth observed on inorganic salts-starch agar; no growth observed ontomato paste-oatmeal agar.

Vegetative Mycelium

On media where good growth occurred, the vegetative mycelium wasobserved to be raised and convoluted and was generally yellowish whitein color. A fetid odor was observed on several media.

Aerial Mycelium and Spore Color

Aerial mycelium absent on most media; very sparse white mycelia whenpresent.

Soluble Pigments

Absent on most media; yellowish pigment on NZ-amine-starch-glucose agar.

Physiological Reactions

No melanin pigments on peptone-iron agar and tyrosine agar (ISP-7);strong peptonization of litmus milk; no proteolysis of nutrient gelatin;weak to no reduction of nitrate, no hydrolysis of tyrosine or xanthine;strong hydrolysis of adenine, and hypoxanthine; weak hydrolysis ofstarch; strong hydrolysis of esculin; no hydrolysis of urea.Carbohydrate utilization as per the method of T. G. Pridham and D.Gottleib. The utilization of carbon compounds by some Actinomycetales asan aid for species determination. J. Bacteriol., 56: 107-114 (1948):good utilization of galactose, glucose, glycerol, lactose, maltose,mannitol, trehalose and xylose; moderate utilization of arabinose,fructose, mannose, rhamnose and sucrose; poor utilization of melibioseand salicin; no utilization of adonitol, dulcitol, inositol, melezitose,raffinose and sorbitol. Acid production from carbohydrates by the methodof R. E. Gordon et al. (vide supra); Good acid production fromarabinose, fructose, galactose, glucose, glycerol, maltose, mannitol,mannose, rhamnose, salicin, sucrose and trehalose; weak acid productionfrom lactose, melezitose and raffinose. Utilization of organic acids bythe method of R. E. Gordon et al. (vide supra); utilization of lactateand malate; no utilization of benzoate, citrate, mucate and oxalate.

                                      TABLE I                                     __________________________________________________________________________    Cultural Characteristics of Glycomyces harbinesis LL-D01539                   Incubation 14 days             Temperature: 28° C.                            Amount of              Soluble                                                                            Reverse                                    Medium Growth                                                                              Aerial Mycelium and/or Spores                                                                  Pigment                                                                            Color                                      __________________________________________________________________________    Benedict's                                                                            Moderate                                                                           Smooth, somewhat plicate                                                                       None 92. yellow-                                agar         vegetative mycelia, 92. yellow-                                                                     ish white                                               ish white; no aerial mycelia or                                               spores; fetid odor                                               Bennett's                                                                            Moderate                                                                            Raised waxy white growth; no                                                                   None 92. yellow-                                agar         aerial mycelia; fetid odor                                                                          ish white                                  Calcium                                                                              Moderate                                                                            White vegetative mycelia with                                                                  None --                                         Malate agar                                                                          to good                                                                             white aerial mycelia                                             Czapek's                                                                             Good to                                                                             White vegetative mycelia with                                                                  None 89. pale-                                  Sucrose                                                                              moderate                                                                            copious white aerial mycelia and                                                                    yellow                                     agar         spores                                                           Gauze No. 1                                                                          Moderate                                                                            Flat, dull colorless vegetative                                                                None --                                         agar         mycelia, aerial mycelia                                          Hickey Moderate                                                                            Plicate, waxy growth, 92.                                                                      None --                                         Tresner      yellowish white, no aerial                                       agar         mycelia; fetid odor                                              Inorganic                                                                            Moderate                                                                            Flat, colorless to white                                                                       None Colorless                                  Salts- to poor                                                                             vegetative growth with sparse                                    Starch agar  white aerial mycelia around edges                                             of colonies                                                      NZ--amine-                                                                           Good  Raised, ridged vegatative growth                                                               yellow-                                                                            --                                         Starch       92. yellowish white, trace white                                                               ish                                             Glucose      aerial mycelia                                                   agar                                                                          Oatmeal                                                                              Moderate                                                                            Flat colorless to white vege-                                                                  None --                                         agar         tative growth with no apparent                                                aerial mycelia                                                   Tomato None                                                                   Paste-Oat-                                                                    meal agar                                                                     Yeast  Good  Plicate, raised growth, 92.                                                                    None 89. pale                                   Extract-     yellowish white; no aerial                                                                          yellow                                     Malt         mycelia                                                          Extract                                                                       agar                                                                          __________________________________________________________________________

                  TABLE II                                                        ______________________________________                                        Micromorphology of Glycomyces harbinesis LL-D05139                                   Aerial Mycelium and/                                                          or Sporiferous Spore           Spore                                   Medium Structures     Shape   Spore Size                                                                            Surface                                 ______________________________________                                        Czapek's                                                                             Rudimentary aerial                                                                           Cylind- 0.50 ± 0.05                                                                        Smooth                                  Sucrose                                                                              sporophores carrying                                                                         rical   micron ×                                  agar   relatively short       1.02 ± 0.04                                         straight chains of     micron                                                 mature spores                                                          ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Physiological Reactions of Glycomyces                                         harbinensis LL-D05139                                                                 Incubation                                                                              Amount of   Physiological                                   Medium  Period    Growth      Reaction                                        ______________________________________                                        Peptone-                                                                               7 Days   Good        No pigment                                      Iron    14 Days   Good        No pigment                                      agar                                                                          Tyrosine                                                                               7 Days   Good        Slight reddish pigment                          agar    14 Days   Good        Slight reddish pigment                          Litmus  14 Days   Good        Strong peptonization                            Milk    28 Days   Good        Strong peptonization                            Nutrient                                                                              14 Days   Good        No proteolysis                                  Gelatin 28 Days   Good        No proteolysis                                  Nitrate 14 Days   Good        No reduction                                    Broth   28 Days   Good        Weak reduction                                  Adenine 14 Days   Good        Hydrolysis                                      Agar    28 Days   Good        Strong hydrolysis                               Hypox-  14 Days   Good        Hydrolysis                                      anthine 28 Days   Good        Strong hydrolysis                               agar                                                                          Tyrosine                                                                              14 Days   Good        No hydrolysis                                   agar    28 Days   Good        No hydrolysis                                   Xanthine                                                                              14 Days   Good        No hydrolysis                                   agar    28 Days   Good        No hydrolysis                                   Urea    28 Days   Good        No decomposition                                broth                                                                         Esculin 14 Days   Good        Strong hydrolysis                               broth                                                                         Starch  14 Days   Good        Weak hydrolysis                                 agar                                                                          ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Carbon Source utilization of Glycomyces harbinensis                           LL-D05139 on ISP-9 Carbohydrate Utilization Medium                            Incubation: 28 Days                                                                           Temperature: 28° C.                                    Carbon Source   Utilization                                                   ______________________________________                                        Adonitol        0                                                             l-Arabinose     2                                                             Dulcitol        0                                                             Fructose        2                                                             d-Galactose     3                                                             d-Glucose       3                                                             Glycerol        3                                                             i-Inositol      0                                                             Lactose         3                                                             Maltose         3                                                             d-Mannitol      3                                                             d-Mannose       2                                                             d-Melezitose    0                                                             d-Melibiose     1                                                             d-Raffinose     0                                                             l-Rhamnose      2                                                             Salicin         1                                                             Sorbitol        0                                                             Sucrose         2                                                             d-Trehalose     3                                                             d-Xylose        3                                                             Negative control                                                                              0                                                             ______________________________________                                         *3 = Good Utilization                                                         *2 = Fair Utilization                                                         1 = Poor Utilization                                                          0 = No Utilization                                                       

                  TABLE V                                                         ______________________________________                                        Acid Production from Various Carbohydrates by                                 Glycomyces harbinesis LL-D05139 on Gordon's                                   Basal Inorganic nitrogen Medium                                                                Temperature: 28° C.                                   Incubation: 28 Days                                                                            Acid Production                                              Carbon Source    7 Days   28 Days                                             ______________________________________                                        Adonitol         -        -                                                   l-Arabinose      +        +                                                   Dulcitol         -        -                                                   Fructose         +        +                                                   d-Galactose      +        +                                                   d-Glucose        +        +                                                   Glycerol         +        +                                                   i-Inositol       -        -                                                   Lactose          ±     ±                                                Maltose          +        +                                                   d-Mannitol       +        +                                                   d-Mannose        +        +                                                   d-Melezitose     -        ±                                                d-Melibiose      -        -                                                   d-Raffinose      -        ±                                                l-Rhamnose       ±     +                                                   Salicin          +        +                                                   Sorbitol         -        -                                                   Sucrose          +        +                                                   d-Trehalose      +        +                                                   d-Xylose         +        -                                                   Negative control -        -                                                   ______________________________________                                         + = Positive response                                                         - = Negative response                                                         ± = Weak Positive Response                                            

                  TABLE VI                                                        ______________________________________                                        Utilization of Organic Acids by Glycomyces harbinensis                        LL-D05139 on Gordon's Modification of Koser's Basal Agar                      (Koser's Citrate Agar)                                                        Incubation: 28 Days                                                                           Temperature: 28° C.                                    Carbon Source   Utilization                                                   ______________________________________                                        Benzoate        -                                                             Citrate         -                                                             Lactate         +                                                             Malate          +                                                             Mucic Acid      -                                                             Oxalate         -                                                             Succinate       -                                                             ______________________________________                                         + = Positive response                                                         - = Negative response                                                    

It is to be understood that for the production of this antibacterialagent the present invention is not limited to this particular organismor to organisms fully answering the above growth and microscopiccharacteristics, which are for illustrative purposes only. In fact, itis desired and intended to include in the term "Glycomyces harbinensisgen. nov., sp. nov., NRRL 15337" the natural (spontaneous) mutants ofthis organism as well as induced mutants produced from this organism byvarious mutagenic means known to those skilled in the art, such asexposure to X-ray radiation, ultraviolet irradiation, nitrogen mustard,actinophages, nitrosamines and the like. It is also desired and intendedto include inter- and intraspecific genetic recombinants produced bygenetic techniques known to those skilled in the art, such as, forexample, conjugation, transduction, and genetic engineering techniques.

This antibacterial agent is active in vitro against gram-positive andgram-negative bacteria when tested by the standard agar dilutionprocedure with Mueller-Hinton agar, and an inoculum of each testorganism of approximately 10⁴ colony-forming units delivered by a Steersreplicating device. The minimal inhibitory concentration (mcg/ml) wasdefined as the lowest concentration of antibiotic LL-D05139β thatinhibited visible growth after 18 hours incubation of 35° C. The resultsare given in Table VII.

                  TABLE VII                                                       ______________________________________                                        In vitro Antibacterial Activity of LL-D05139β                                               Minimal Inhibitory                                         Organism (Name & Number)                                                                         Concentration (mcg/ml)                                     ______________________________________                                        Escherichia coli                                                                          Stfd-79-20  8                                                      "          #31         4                                                      "          ATCC 25922  8                                                     Klebsiella  Stfd-79-6  128                                                    pneumoniae                                                                    Klebsiella  SSC-78-1   128                                                    pneumoniae  AD          4                                                     Enterobacter                                                                              Stfd-79-14 128                                                    aerogenes                                                                     Enterobacter                                                                              K-79-16    128                                                    cloacae                                                                       Serratia sp TU1-78-15  64                                                     Serratia    QHC-77-2   32                                                     marcescens                                                                    Proteus morganii                                                                          K-79-25    64                                                     Proteus rettgeri                                                                          N-76-1     16                                                     Providencia SSC-80-78  64                                                     stuarti                                                                       Providencia K-81-29    64                                                     stuarti                                                                       Salmonella sp.                                                                            QHC-77-3    4                                                     Pasteurella LL #117    64                                                     multocida                                                                     Acinetobacter                                                                             Stfd-79-17 512                                                    calcoaceticus                                                                 Acinetobacter                                                                             K-77-1     128                                                    calcoaceticus                                                                 Pseudomonas SSC-78-13  512                                                    aeruginosa                                                                    Pseudomonas 12-4-4     128                                                    aeruginosa                                                                    Pseudomonas ATCC 27853 128                                                    aeruginosa                                                                    Staphylococcus                                                                            SSC-79-18  16                                                     aureus                                                                        Staphylococcus                                                                            FU-79-2    16                                                     aureus                                                                        Staphylococcus                                                                            ATCC 25923 16                                                     aureus                                                                        Enterococcus sp.                                                                          OSU-75-1   64                                                     Enterococcus sp.                                                                          SM-77-15   64                                                     Micrococcus PCI-1001    4                                                     luteus                                                                        Bacillus subtilis                                                                         ATCC 6633  16                                                     Candida albicans                                                                          LL #32     64                                                     Candida albicans                                                                          LL #54     128                                                    ______________________________________                                    

In addition LL-D05139β inhibits the growth of transplanted mouse tumorsas established by the following test.

Lymphocytic leukemia P388 test

The animals used were DBA/2 mice, all of one sex, weighing a minimum of17 g and all within a 3 g weight range. There were three animals pertest group. The tumor transplant was by intraperitoneal injection of 0.1ml of dilute ascitic fluid containing 10⁶ cells of lymphocytic leukemiaP388. LL-D05139β was administered intraperitoneally on days 1,5 and 9(relative to tumor inoculation) at various doses. The animals wereweighed and survivors recorded on a regular basis for 30 days. The ratioof survival time for treated (T)/control (C) animals was calculated. Thepositive control compound was 5-fluorouracil, given as a 60 mg/kginjection. The criterion for efficacy is T/C×100≧125%. The results ofthis test appear in Table VIII.

                  TABLE VIII                                                      ______________________________________                                        Lymphocytic Leukemia P388 Test                                                Compound      Dose mg/kg T/C × 100(%)                                   ______________________________________                                        LL-D05139β                                                                             200        140                                                                50         150                                                                25         140                                                  ______________________________________                                    

Further LL-D05139β was tested in the human tumor clonogenic (stem cell)assay and found to be active against both the human lung (LX-1)carcinoma and the colon(CX-1) carcinoma at 2.0 mcg/ml and higher doses.

Human Tumor Clonogenic Assay

This test was performed essentially by the method of A. Hamburger and S.Salmon, Primary bioassay of human tumor stem cells., Science, 197:461-463 (1977). Human colon carcinoma (CX-1) and human lung carcinoma(LX-1) tumors were obtained from the National Cancer Institute andpropagated in athymic Balb/c mice. Freshly removed tumors were dispersedwith trypsin 0.25% and 5×10⁴ lung carcinoma cells or 1×10⁵ coloncarcinoma cells in soft (0.3%) agarose medium containing 0.1 ml of testdrug were plated onto a solidified (0.5%) agarose base medium in 35 mmgrided dishes. Cultures were incubated at 37° C. in a humidified 5%carbon dioxide in air atmosphere. Colonies were counted after 10-11days. A compound is considered active if the treated/control (T/C) valueis ≦30%, representing a 70% cell kill. The results of this test onLL-D05139β appear in Table IX.

                  TABLE IX                                                        ______________________________________                                        Effect of LL-D05139β on Human Lung and Colon Carcinoma.                  Clonogenic Assay                                                                           Lung Carcinoma                                                                           Colon Carcinoma                                                Concen-   Av. No. of     Av. No. of                                           tration   Colonies/ T/C  Colonies/                                                                             T/C                                 Compound (mgc/ml)  Culture   (%)  Culture (%)                                 ______________________________________                                        Control  0         590            420                                         LL-D05139β                                                                        200        78       13.0                                                      20        105       17.8                                                      2         137       23.2  67     16.0                                ______________________________________                                    

General Procedure for the Production of LL-D05139β

Cultivation of Glycomyces harbinensis NRRL 15337 may be carried out in awide variety of liquid culture media. Media which are useful for theproduction of LL-D05139β include an assimilable source of carbon such asstarch, sugar, molasses, glycerol, etc., an assimilable source ofnitrogen such as protein, protein hydrolysates, polypeptides, aminoacids, corn steep liquor, etc., and inorganic anion and cation saltssuch as potassium, sodium, ammonium, calcium, sulfate, carbonate,phosphate, chloride, etc. Trace elements such as boron, molybdenum,copper, etc. are supplied as impurities of other constituents of themedia. Aeration in tanks, bottles and flasks is provided by forcingsterile air through or onto the surface of the fermenting medium.Agitation is supplied by a mechanical impeller. An antifoaming agentsuch as lard oil or silicone defoamer may be used as needed.

General Procedure for the Isolation of LL-D05139β

Due to its poor stability in acid, solutions containing LL-D05139β arekept between pH 7 and pH 9.5 throughout the isolation processes. Whendefined medium is used for the fermentation, only a trace amount ofazaserine is coproduced, LL-D05139β can be recovered from thefermentation by adsorption of the fermentation filtrate on a column ofgranular carbon and elution from the column by aqueous alcohol mixture.The active preparation is further purified by repeated columnchromatography on microcrystalline cellulose, eluting with1-propanol:water (80:20). When complex medium is used for thefermentation, due to the presence of a large amount of interferingimpurities, LL-D05139β cannot be adsorbed from the fermentation filtrateby granular carbon. In this case, the fermentation filtrate is passedthrough a column of granular carbon to remove azaserine and otherimpurities. LL-D05139β in the column effluent is then adsorbed onto weakanion-exchange resins such as Amberlite® IR45(OH.sup.⊖ oranion-exchanger such as DEAE Sephadex® or QAE Sephadex®. The activitycan be eluted from the resin or Sephadex® with dilute aqueous basefollowed by desalting on a granular carbon column. Antibiotic LL-D05139βis then eluted from the granular carbon column with aqueous alcoholmixtures and further purified as described before.

At the end of each chromatographic step, the partially purifiedpreparations can be conveniently quantitated against analytically pureLL-D05139β using the following HPLC system:

Column: Nucleosil 10, N(CH₃)₂, 25 cm (Macherey-Nagel packed column).

Solvent: Methanol:2-propanol:water, 75:5:20.

Detector: 254 nm.

Flow rate: 1.5 ml/minute.

Retention Volume of LL-D05139β=6 ml.

Also embraced within the purview of the present invention aretherapeutic compositions of matter useful for treating bacterialinfections and ameliorating cancer diseases in mammals which contain thenovel antibiotic of the present invention as the active ingredientthereof. This aspect of the invention includes the novel compositions ofmatter and the method of treating bacterial infections in mammals whenadministered in amounts of approximately 100 mg to approximately 10 gper day for a 70 kg subject. The daily dosage may be given as divideddoses several times a day. The dosage range is to be adjusted to provideoptimum therapeutic response in the mammal being treated.

The instant invention further includes the novel compositions of matterand the method of inducing the regression and/or palliation of leukemiaand related cancers in mammals when administered in amounts ranging fromabout 0.075 mg to about 300 mg per square meter of body surface area perday. The interrelationship of dosages for animals of various sizes andspecies and humans (based on mg/m² of surface area) is described byFreireich, E. J., et al., Quantitative Comparison of Toxicity ofAnticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man, CancerChemother. Rep., 50 No. 4: 219-244 (May 1966). A preferred dosageregimen for optimum results would be a total treatment dose from about1.0 mg/m² to about 8 mg/m². Such dosage units are employed that a totalof from about 0.5 mg to about 525 mg of the active compound for asubject of about 70 kg of body weight are administered in a 24 hourperiod. This dosage regimen may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The activecompound may be specifically administered for the anticancer use by theintravenous, intramuscular, or subcutaneous routes.

Regression and palliation of cancers are attained, for example, usingintraperitoneal administration. A single intravenous dosage or repeateddaily dosages can be administered. Daily dosages up to about 5 to 10days are often sufficient. It is also possible to dispense one dailydosage or one dose on alternate or less frequent days. As can be seenfrom the dosage regimens, the amount of principal active ingredientadministered is a sufficient amount to aid regression and palliation ofthe leukemia or the like, in the absence of excessive deleterious sideeffects of a cytotoxic nature to the hosts harboring the cancer. As usedherein, cancer disease means blood malignancies such as leukemia, aswell as other solid and non-solid malignancies such as themelanocarcinomas, lung carcinomas, and mammary tumors. By regression andpalliation is meant arresting or retarding the growth of the tumor orother manifestation of the disease compared to the course of the diseasein the absence of treatment.

The active compounds may be administered parenterally orintraperitoneally. Solutions or dispersions of the active compound canbe prepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount on the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In therapeutic use, the compounds of this invention may also beadministered in the form of conventional oral pharmaceuticalcompositions. Such compositions may be suitable for oral administrationwhen formulated properly so the compound of the present invention is notexposed to the acid pH of the stomach. The active ingredient may becombined in admixture with a pharmaceutically acceptable carrier, whichcarrier may take a wide variety of forms depending on the form ofpreparation desired for administration. The compounds can be used incompositions such as tablets. Here, the principal active ingredient ismixed with conventional tabletting ingredients such as corn starch,lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,dicalcium phosphate, gums, or similar materials as nontoxicpharmaceutically acceptable diluents or carriers. The tablets or pillsof the novel compositions can be laminated or otherwise compounded toprovide a dosage form affording the advantage of prolonged or delayedaction or predetermined successive action of the enclosed medication.For example, the tablet or pill can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids ormixtures of polymeric acids with such materials as shellac, shellac andcetyl alcohol, cellulose acetate and the like. A particularlyadvantageous enteric coating comprises a styrene maleic acid copolymertogether with known materials contributing to the enteric properties ofthe coating. The tablet or pill may be colored through the use of anappropriate nontoxic dye, so as to provide a pleasing appearance. Inaccord with this invention, another example of a suitable oral dosageform is the capsule with the active ingredient specifically formulatedto be absorbed in the duodenum.

It is especially advantageous to formulate parenteral and oralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activematerial and the particular therapeutic effect to be achieved, or (b)the limitations inherent in the art of compounding such an activematerial for the treatment of disease in living subjects having adiseased condition in which bodily health is impaired as hereindisclosed in detail.

The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically-acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from about 0.1 to about 500 mg., withfrom about 10 to about 500 mg. being preferred. Expressed in proportionsfor parenteral use, the active compound is generally present in fromabout 0.1 to about 100 mg/ml of carrier. In the case of compositionscontaining supplementary active ingredients, the dosages are determinedby reference to the usual dose and manner of administration of the saidingredients.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatable with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

This invention will be described in greater detail in conjunction withthe following non-limiting examples.

EXAMPLE 1 Inoculum Preparation

A typical medium used to grow the primary inoculum was preparedaccording to the following formula:

    ______________________________________                                        Dextrose         1.0%                                                         Starch           2.0%                                                         Yeast extract    0.5%                                                         N--Z Amine       0.5%                                                         Calcium carbonate                                                                              0.1%                                                         Water qs         100%                                                         ______________________________________                                    

The pH was adjusted to 7.2 with sodium hydroxide and the medium wassterilized. Mycelial scrapings from an agar slant of Glycomycesharbinensis gen. nov., sp. nov., NRRL 15337 were used to inoculate 50 mlof the above sterile medium in a 250 ml flask. The flask was incubatedon a rotary shaker at 28° C. and 180-200 rpm for 72 hours. The resultingprimary inoculum was used to inoculate a series of 500 ml flasks, eachcontaining 100 ml of the above sterile medium, which were then incubatedunder the same conditions, providing secondary inoculum.

EXAMPLE 2 Fermentation

A fermentation medium was prepared according to the following formula:

    ______________________________________                                        Dextrose          2.0%                                                        Sodium Nitrate    0.1%                                                        Ferrous Sulfate   0.01%                                                       heptahydrate                                                                  Magnesium sulfate 0.02%                                                       heptahydrate                                                                  Calcium carbonate 0.5%                                                        Water qs          100%                                                        ______________________________________                                    

The pH was adjusted to 7.4 and the medium was sterilized. Portions ofsecondary inoculum (Example 1) was used to inoculate 9 liters of theabove sterile medium, which was then incubated on a rotary shaker at180-200 rpm and 28° C. for 6 days at which time the mash was harvested.

EXAMPLE 3 Isolation of LL-D05139β

A 9 liter portion of harvest mash (prepared as described in Example 2)was filtered. The 8200 ml of filtrate (containing 164 ug/ml ofLL-D05139β as determined by HPLC) was adjusted to pH 9.0 with theaddition of dilute aqueous sodium hydroxide. The filtrate was thencooled in an ice-water bath while being passed through a column packedwith 700 ml of activated charcoal, at a flow rate of 16 ml/minute. Thecolumn was washed with 700 ml of demineralized water and then elutedwith a linear gradient from water to a mixture of water:methanol (4:6)over a period of 6 hours at the rate of 2.6 ml/minute, collecting 5minute fractions. The elution was continued until 120 fractions werecollected. Each fraction was analyzed by thin-layer chromatography(TLC)(with silica gel 60, F254, precoated TLC sheets, product of E.Merck Inc.; n-propanol:water (80:20); visualized by F254 quenching andninhydrin spray; Rf=0.27) and agar diffusion assay against Escherichiacoli. Fractions 7-57, 58-93 and 94-118 were pooled separately and eachpool was concentrated in vacuo to remove the methanol and was thenneutralized with dilute hydrochloric acid and freeze-dried, givingrespectively 1427 mg (A), 423 mg (B) and 148 mg (C) of yellow solids.

A 748 mg portion of (A) was dissolved in 1.5 ml of water, loaded on aglass column (2.5×110 cm) packed with microcrystalline cellulose andequilibrated in n-propanol:water (80:20). The column was eluted withn-propanol:water (80:20) at a flow rate of 2 ml/minute, collecting 6minute fractions to a total of 120 fractions. Each fraction was analyzedas described above. Fractions 80-93 and 94-102 were pooled separately.Each pool was azeotroped with water (1/2 volume) in vacuo, neutralizedwith dilute hydrochloric acid and freeze-dried, giving respectively 189mg of white powder (D) and 58 mg of pale yellow solids (E). Sample (E)was analytically pure LL-D05139β as determined by high pressure liquidchromatography (HPLC) and TLC. Sample (D) contained 48% LL-D05139β byHPLC.

A second portion of (A) (676 mg) was chromatographed as described above,giving 176 mg of white powder (F) which was 34% LL-D05139β and 83 mg ofpale yellow solids (G), which was 89% LL-D05139β.

Samples (B) (420 mg), (C) (147 mg) and 600 mg of product derived fromanother fermentation conducted as described in Example 2, were combinedand chromatographed as described above, giving 262 mg of white powder(H) which was 33% LL-D05139β and 63 mg of pale yellow solids (J) whichwas 82% LL-D05139β.

Products (D), (F) and (H) were combined and chromatographed on a columnof microcrystalline cellulose in the same manner as described above,giving 402 mg of white powder (K) which was 27% LL-D05139β and 80 mg ofpale yellow solids (L) which was 100% LL-D05139β.

The analytical data for (L) were as follows:

Elemental analysis: C36.8; H,5.2; N,19.6;

Molecular weight: 244 (FAB-MS);

Optical Rotation: [α]_(D) ²⁶ =+57°±5°(C,0.19%, Water);

UV_(max) 250 nm (E_(1cm) ^(1%) 646) (Spectrum as shown in FIG. I,methanol/water solution);

IR spectrum (KBr disc) as shown in FIG. II;

¹ H NMR spectrum as shown in FIG. III (D₂ O; ppm from TMS) 1.56(3H, d,J=7.4 Hz), 4.07(1H, q, J=7.4 Hz), 4.49(2-3H, m), 5.19(1H, broad s).

We claim:
 1. A process for preparing antibiotic LL-D05139β whichcomprises cultivating Glycomyces harbinensis, gen. nov., sp. nov.,having the identifying characteristics of NRRL 15337, and theantibiotic-producing mutants thereof, under areobic conditions, in asterile liquid medium containing assimilable sources of carbon, nitrogenand inorganic anion and cation salts, until substantial antibioticactivity is imparted to said medium by the production of LL-D05139β andthen recovering the antibiotic therefrom.
 2. A process for preparingantibiotic LL-D05139β which comprises aerobically fermenting a liquidmedium containing assimilable sources of carbon, nitrogen and inorganicanion and cation salts, which medium has been inoculated with a viableculture of the organism Glycomyces harbinensis, gen. nov., sp. nov.,having the identifying characteristics of NRRL 15337, and theantibiotic-producing mutants thereof, maintaining said fermentationculture with sterile aeration and agitation at pH 7-9.5, for a period of90-200 hours at 24°-32° C., harvesting the mash and extracting theantibiotic.
 3. The process according to claim 2, wherein the pH of theliquid medium is 7.4.
 4. A biologically pure culture of themicroorganism Glycomyces harbinensis, gen. nov., sp. nov., having theidentifying characteristics of NRRL 15337, said culture being capable ofproducing the antibiotic LL-D05139β in recoverable quantity uponfermentation in an aqueous nutrient medium containing assimilablesources of carbon, nitrogen and inorganic anion and cation salts.
 5. Thebiologically pure culture of the microorganism Glycomyces harbinensisaccording to claim 4, wherein said microorganism has spontaneouslymutated, such that the microorganism is genetically altered but stillretains the ability to synthesize antibiotic LL-D05139β.
 6. Thebiologically pure culture of the microorganism Glycomyces harbinensisaccording to claim 4, wherein said microorganism has been subjected tomutagenic means such that the microorganism is genetically altered butstill retains the ability to synthesize antibiotic LL-D05139β.